Prolong diamond antifade mountant with 4 6 diamidino 2 phenylindol dapi

Cytospins of NALM6 alone and co-cuture samples of NALM6 with BM-MSC were fixed with 4% paraformaldehyde and washed with PBS, followed by 1 h incubation in a 3% BSA blocking buffer. After removal of blocking buffer, Cells were permeabilized by incubation with 0.1% Triton X-100 in PBS for 10 min at room temperature and then incubated for 1 h with primary anti-ITGB1 antibody (P5D2, Abcam) diluted in 3% NGS/0.1% Triton X-100/PBS. Slides were washed three times in PBS, and then incubated for 1 h in 3% NGS/0.1% Triton X-100/PBS containing goat anti-rabbit Alexa Fluor 568 (Invitrogen, Carlsbad, CA). Slides were washed three times in PBS and mounted with ProLong Diamond Antifade Mountant with 4′-6-diamidino-2-phenylindol (DAPI) (ThermoFisher Scientific). All steps were carried out at room temperature. Images were captured using a Nikon C2 confocal fluorescence microscope and analyzed using NIS Elements software.